Study Title:

Chlorella Enhances Dendritic Cell Immune Function

Study Abstract

Chlorella sorokiniana (CS) is a unicellular green alga. The extracts of Chlorella have been used as treatments for relieving hypertension and modulating immune response. The detailed mechanisms are not clear yet. In this study, we sought to study the molecular mechanisms for the polysaccharide fraction of CS-induced immune response. We pulsed dendritic cells (DCs) with CS and found that CS could maturate DCs. CS-maturated DC could activate naïve T cells and stimulate T-cell proliferation and IFN-γ secretion. Furthermore, CS activated PI3K and MAPKs signaling pathways in DCs by interacting with TLR4 receptor. These CS-activated signaling pathways could further activate NF-κB and induce IL-12 production in DCs. This study provides molecular mechanisms for CS-induced DCs activation and immune response.

Introduction .

Chlorella is a genus of freshwater unicellular green algae. The extracts of Chlorella have been proposed as potential treatments for improving human health and wildly used as botanical foods. For example, Chlorella extracts were used as nutrition supplements in relieving hypertension and treatments for modulating human immune responses [1–4]. It was reported that Chlorella extracts can elicit various beneficial pharmacological effects against cancers [5], bacterial infections [6], and viral replication [7, 8]. From an earlier study, Chlorella extracts were shown to strongly increase the production of IFN-γ and IL-2 and activate Th1 cells to strengthen immune system and host defense [9]. Hasegawa et al. demonstrated the roles of Chlorella extracts in inducing IFN-γ and IL-2 mRNA expression and activating cell-mediated immunity [10].

DCs are professional antigen-presenting cells (APCs) and have unique ability in linking innate and adaptive immunity [11, 12]. Immature DCs are able to ingest antigens. Once activated, DCs go through a series of maturation processes that include migration to lymphoid tissues, downregulation of antigen uptake, upregulation of major histocompatibility complex (MHC) class II, costimulatory molecules (CD40, CD80, and CD86), and a specific maturation marker CD83 [13–15], and finally presenting antigenic peptides to T lymphocytes [16].

The MAPK families (p38, ERK, and JNK) were activated in response to a variety of cellular stress or stimuli, including oxidative stress, LPS and TNF-α, in all cell types indicated [17, 18].

The effects of CS on human DCs are not yet defined. In this study, we examined the molecular mechanisms of CS in the activation and maturation processes of human monocyte-derived DC.

Discussion .

Algae are simple organisms, and their extracts can regulate immune responses in mammals [33, 34]. Among these algae, several species of Chlorella were reported to induce immune responses in human or mice [35–37]. In this study, we studied the effect of Chlorella sorokiniana polysaccharides extracts on human immune responses. It was reported that polysaccharides extracted from several origins can induce DC maturation and immune responses [38]. As other polysaccharide extracts, Chlorella polysaccharide fraction extracts can also maturate DC and activate immune response by inducing IL-12 secretion in DCs.

IL-12 is important for activating natural killer cells (NKs) and inducing the differentiation of T helper cells toward Th1 cells. Th1 response can skew the immune system toward cellular immune response, which maximize the killing efficacy of macrophage, increase CD8+ T-cell proliferation, and activate natural killer cells [39]. In addition, Th1 response is important in fighting against virus infection and cancer [40].

Since NF-κB binding site was found in the promoter region of both IL-12 p40 and p35, NF-κB may involve in CS-induced IL-12 expression. Indeed, we observed that CS not only induced the expression of IL-12 in DC but also the activation of NF-κB. Furthermore, blocking NF-κB signaling in DCs reduced CS-induced IL-12 secretion. Therefore, NF-κB signaling is required for CS-induced IL-12 expression in DCs.

Signal transductions via the MAPKs play important roles in cellular responses including cell proliferation, differentiation, and survival [41]. We detected the activation of AKT and MAPK signaling pathways induced by CS in DC and verified the importance of AKT and MAPK signaling pathways induced by CS in DC activation. All signaling pathways tested were required for CS-induced IL-12 secretion in DC. However, ERK signaling pathway is not required for CS-induced DC maturation. The role of ERK signaling pathway in DC maturation is reported controversially. Lin et al. reported that ERK signaling pathway is required for IL-12 secretion in DC maturated by polysaccharide purified from Ganoderma lucidum [4]. However, ERK pathway was shown to differentiate DC towards tolerogenic DC [42]. One possible explanation is that the intensity and duration of signaling activation may be different based on stimuli, and different activation patterns of signaling pathways may result in various consequences. Further studies are required to clarify the importance of the intensity and duration of ERK phosphorylation in DC activation.

TLRs are cell surface receptors that recognize structurally conserved molecules derived from microbes. The reported ligands of these TLRs range from bacterial cell surface LPS, lipoproteins, flagellin, viral DNA and dsRNA to host fibrinogen and heat shock proteins. Our results suggested that CS interacts with TLR4 and maturates DC through NF-κB signaling pathway. Interestingly, TLR4 activates genes required for initiating adaptive immune responses when artificially ligated by antibodies, and NF-κB signaling pathway was reported involved in this process [43]. Consistent with previous publications, we demonstrated the importance of TLR4 signaling in activating immune response. Furthermore, we identified CS extracts as novel ligands for TLR4. In order to rule out the contamination of endotoxin LPS in CS, we detected LPS level in CS samples. We found that there was no detectable level of endotoxin (<0.30 endotoxin units/mL) in CS samples (data not shown). Therefore, our results suggested that CS maturates DC by binding to TLR4 receptor.

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